Viruses, which are known to cause human disease (e.g., colds, influenza, polio, AIDS, etc.), are not considered live cells in the traditional sense but infect living cells where they can replicate. Viruses may exist in the living cells used to produce biopharmaceutical products, thereby presenting a serious risk of contaminating the biopharmaceutical products during processing. In addition, viruses can be introduced into biopharmaceutical products through reagents fed to the process (especially of concern is cell culture media including components derived from animal origin) and through accidental exposure to the processing environment or personnel. Such contaminating viruses must be removed or inactivated prior to (e.g., in the case of inactivating viruses in the cell culture media prior to introduction in the facility or process), during, or after the manufacturing or processing of the product without harming or destroying the often delicate biopharmaceutical or other sensitive product.
In addition to biopharmaceutical and biologically derived products, there are many other sensitive products including synthetic media, blood, cell therapy, protein, small molecule pharmaceuticals, nutrition products, infant formula, liquid or non-liquid form of all, that must be manufactured and delivered free of active viruses.
For many biopharmaceutical products, the United States Food and Drug Administration (FDA) requires less than one virus per million doses of product sold. To achieve this target, current virus control strategies used in biopharmaceutical manufacturing operations often utilize multiple inactivation techniques. One of the most common techniques is virus filtration, generally using direct flow filtration. Another common method is low pH inactivation, in which the solution containing the biopharmaceutical product is held in a vessel and treated with acid. The efficacy of inactivation depends not only on the sensitivity of the virus to the acidic conditions, but also on the sensitivity of the biopharmaceutical product. The pH level and time of exposure to the acidic conditions are chosen so as to minimize any damage to the resulting biopharmaceutical product.
Chromatography is the most common technique for purifying the desired biopharmaceutical product from other synthesis by-products or wastes, and biopharmaceutical manufacturers leverage their chromatography steps to further augment their virus safety assurance. For example, it has been shown that a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling (e.g., sodium thiocyanate or urea), can significantly inactivate viruses.
Other chemical treatments have also been shown to be effective for virus inactivation. For example, solvent/detergent (S/D) treatment is commonly used to inactivate enveloped viruses by disrupting the lipid envelope surrounding the viruses. Chemical treatments may necessitate additional purification and filtration processing to remove the chemicals from the product solution after virus inactivation is complete.
High temperature pasteurization can also be effective for inactivating viruses, although additional stabilizers are often required in the product solution to help the biopharmaceutical product withstand pasteurization conditions. In the case of high temperature, short time viral inactivation sometimes used on cell culture media, specific components are degraded by the process and must be removed, then added back to the media after processing, reducing the potential efficacy of the viral inactivation.
Other contemplated means for virus inactivation involve the use of carbon dioxide at very high pressures, including at or near supercritical conditions. See, for example, U.S. Pat. No. 5,877,005 (Castor et al.).
There has been extensive work to quantify the effects of carbon dioxide on biological materials, and in particular microbes, spores, and proteins. While the effects of using carbon dioxide against microbes, spores, and proteins are clear, the mechanisms of action remain poorly defined or unknown.
Since microbes are metabolically active, they have ways to combat or defend against the damaging effects of carbon dioxide (e.g., permeability barriers and active transport processes). These microbe defenses must be overwhelmed before killing effects of carbon dioxide are observed. For this reason, the use of carbon dioxide against microbes is generally accomplished at high pressures of about 30 MPa where the carbon dioxide can rapidly cross the cell membrane and accumulate to high intracellular levels.
Use of high pressure carbon dioxide also has been shown to be effective for inactivating spores that are metabolically inert. High pressure carbon dioxide has also been used to inactivate proteins at temperatures where thermal inactivation is ineffective.
However, use of high pressure carbon dioxide that is at or near its supercritical state may not be suitable for virus inactivation during biopharmaceutical manufacturing or the manufacturing and processing of other sensitive products as the high pressure carbon dioxide may damage the biopharmaceutical or other sensitive products. Furthermore, expensive equipment is required to handle carbon dioxide that is at or near its supercritical state, which often renders such treatment processes economically infeasible.
As shown in FIG. 1, a common virus control strategy may typically involve both lowering the pH of the product solution followed by one or more purification and filtration steps to inactivate or remove active virus contaminants. As the variety of potential virus contaminants is great, the use of multiple virus removal or inactivation techniques with different modes of action is highly desirable to ensure unexpected or untested virus threats will be mitigated. In some cases, manufacturers are hindered in optimizing their downstream operations to eliminate un-necessary purification steps, because those steps contribute to their overall virus safety assurance. The selection of virus inactivation techniques for a given biopharmaceutical manufacturing process generally reflects a compromise between the desire to achieve high product yield at reasonable cost versus the need to reduce the risk of virus contamination to acceptably low levels and provide a safe product.
What is needed therefore is an economical method of virus inactivation before, during and/or after biopharmaceutical or other sensitive product manufacturing that is effective for inactivating viruses, provides an orthogonal mode of action to enhance the overall robustness of the virus inactivation strategy, and minimizes any damage or destruction of the biopharmaceutical or sensitive products.